The audiotape was broadcast by the pan-Arab satellite channel Al-Jazeera.
The speaker, who sounded like al-Zawahri on previous audio and videotapes, called on Pakistani soldiers to disobey Musharraf.
"Musharraf seeks to stab the Islamic resistance in Afghanistan in the back," the voice said.
"Every Muslim in Pakistan should work hard to get rid of this agent government, which will continue to submit to America until it destroys Pakistan," the speaker said.
The release of the tape comes amid increased speculation that al-Zawahri escaped a major Pakistani operation against suspected al Qaeda fighters and local sympathizers in its tribal areas bordering Afghanistan.
Musharraf had spoken hopefully of capturing a "high value target" in the rugged border area where local tribes have more power than the Pakistani federal government.
Brig. Mahmood Shah, chief of security in Pakistan's tribal areas, said Thursday that authorities were interrogating 163 captured suspects, but were yet to determine their identities. He acknowledged that some terrorists might have escaped at the start of the operation.
Interior Minister Faisal Saleh Hayyat vowed that the operation in South Waziristan, which began 10 days ago, would continue until the "complete elimination" of terrorists holed up there.
"Over 20 terrorists have been killed in the operation so far and it is expected that 30 to 35 more dead bodies of terrorists will be recovered as the operation concludes," Hayyat told lawmakers in a National Assembly debate. He did not identify the terrorists or say whether they were foreigners or local tribesmen.
In the National Assembly, opposition lawmakers chanted slogans and staged a walkout to protest the operation, the largest since Pakistan threw its support behind the U.S.-led war on terrorism in late 2001. Lawmakers protested the government had not "taken Parliament into confidence" over the operation.
"We have plunged into a such a war which has no end," said Qazi Hussain Ahmed, the leader of the hardline religious coalition Mutahida Majlis-e-Amal. "The whole country is in the grip of uncertainty."
CBS News Analyst Jere van Dyk says he believes the voice on the tape is that of al-Zawahiri.
"The tape touches every single button in Pakistan, particularly in the Pakistani military," he says. "It was an attempt by al-Zawahiri to declare war against Musharraf and to try to divide his military from his officer corps and him in particular.
"The potential for a civil war is great, al-Zawahari knows it."
On Thursday, a 30-member delegation of tribal elders were on a peace mission in the battle zone, near the main South Waziristan town of Wana, seeking the release of 14 Pakistani troops and officials taken captive by hundreds of militants who have been fighting thousands of army forces.
The elders, who left on their mission on Wednesday, were also trying to convince local supporters of al Qaeda to turn over foreign terrorists to the government to avoid a "massive military onslaught," residents and officials said.
Army spokesman Gen. Shaukat Sultan said he had no information on whether the mission had succeeded. "We have not received any response from them," he said.
Pakistan's military had set a Thursday 10 a.m. deadline for the al Qaeda fugitives to surrender themselves and free the hostages. However, there was no fighting reported in the area immediately after the deadline passed, and Shah said the deadline could be extended.
He also told GEO television network that the government was expecting a positive response from the tribal elders. "People should not take these deadlines so seriously. We can extend it," he said.
It was not clear what authorities would do if their demands were not met, although a senior military official involved in the operation said on condition of anonymity that the army would start "a massive onslaught."
The operation in South Waziristan has angered local tribesmen who resent the military's presence in the region. At least two dozen civilians are believed to have died in army firing on vehicles. The military has declined to give full details about its casualties, but officials says at least 30 soldiers have been killed.
The United States has offered a $25 million reward for information leading to the capture of al-Zawahri, a 52-year-old former Egyptian surgeon believed to be the brains behind al Qaeda.
Al-Zawahri's Egyptian Islamic Jihad was believed behind the assassination of President Anwar Sadat during a Cairo military parade in 1981. He merged the organization with al Qaeda in 1998.
CBS National Security Correspondent David Martin reports experts who have researched al-Zawahri's life say he was a
Often seen by Osama bin Laden's side in videos released to Arab television networks, the doctor was also thought to serve as al Qaeda leader's personal physician.